Mesenchymal stromal cells (MSC) display several mechanisms of action that may be harnessed for therapeutic purposes. One of their most attractive features is their immunomodulatory activity that has been extensively characterized both in vitro and in vivo. While this activity has proven to be very efficient, it is transient. We aimed to enhance it by transforming MSC to overexpress a first apoptosis signal (Fas) ligand (FasL). In this study, our goal was to induce FasL overexpression through adenoviral transduction in MSC to improve their immunomodulatory activity. We characterized the impact of FasL overexpression on the morphology, proliferation, viability, phenotype, multilineage differentiation potential and immunomodulation of MSC. Moreover, we determined their suppressive properties in mixed reactions with A20 cells, as well as with stimulated splenocytes. Our findings demonstrate that FasL-overexpressing MSC exhibit improved immunosuppressive properties, while maintaining their MSC-characteristic features. In conclusion, we establish, in a proof-of-concept set-up, that FasL-overexpressing MSC represent good candidates for therapeutic intervention targeted at autoimmune disorders. Mesenchymal stromal cells (MSC) display several mechanisms of action that may be exploited for therapeutic purposes . One of MSC can be sourced from multiple tissues and organs, such as bone marrow (BM), placenta, adipose tissue, umbilical cord blood and many others. MSC have been defined as fibroblast-like nonhematopoietic progenitor cells with the capacity to differentiate in adipogenic, osteogenic and chondrogenic lineages ,13,14,15The first apoptosis signal ligand (FasL)/Fas interaction embodies the typical caspases cascade signaling pathway involved in the apoptosis of many cell types ,20,21. TBM-MSC (from here on referred as MSC) express low levels of FasL and induce cell apoptosis ,9,24. MoTo overcome this inconvenience, we proposed to increase the efficacy of MSC by overexpressing the FasL following adenovirus-mediated expression, thus obtaining “killer” MSC. We chose the adenovirus-mediated transduction of the MSC as it is more suitable to infect slow-cycling cells, like MSC . MoreoveWe report here that the characteristic features of MSC were not affected by FasL overexpression, and their immunosuppressive properties were enhanced, becoming more efficient in inducing the death of both A20 cells and stimulated splenocytes.For the expression of FasL, a truncated sequence of the gene was inserted in the adenoviral expression vector under control of the cytomegalovirus (CMV) promoter alongside green fluorescent protein (GFP) as a reporter protein A,B. The The MSC grown from the bone marrow of nonobese diabetic (NOD) mice displayed a slightly different morphology with a flatter and widespread structure than the regular fibroblast-like, spindle-shaped appearance of MSC derived from wild-type mice, as previously described ,30. The To determine the impact of adenoviral infection, we assessed several specific characteristics of MSC isolated from the bone marrow of NOD mice. First, we evaluated the effect of MSC transduction with FasL on the cell viability. We found that the majority of the naïve bone marrow-derived MSC expressed low levels of Fas ; howeverSecond, the quantification of the doubling time of MSC in culture showed a negligible effect of either GFP or FasL expression, which was around 50 h C. Therefp < 0.005, 2.72 ± 1.08), and the inhibition increased at higher ratios . Effector MSC were co-incubated with CD3/CD28-stimulated splenocyte responders. The proliferation of gated CD8 T cells within the splenocyte preparation as assessed with a dilutional dye increased from 1.16 ± 0.09 to 5.93 ± 2.23 upon CD3/CD28 stimulation . The co-r ratios . Comparar ratios , though r ratios .Altogether, these data indicate that MSC transduction to express either GFP or FasL did not significantly affect the viability, phenotype, cycling rates, differentiation potential or the suppression capacity of these cells.The overexpression of the FasL protein in MSC aims to endow these cells with killing activity. This property was evaluated in cocultures using, as targets, apoptosis-sensitive murine A20 leukemia/lymphoma cells, which express high levels of the Fas receptor A. The A2The efficacy of killer MSC overexpressing FasL in therapeutically relevant settings was tested by a coincubation for 72 h with CD3/CD28-stimulated splenocytes isolated from NOD mice . T cellsMesenchymal stromal cells are endowed with great therapeutic potential, particularly in the inhibition of aberrant immune reactions. A potential use as immune suppressors raises the question of the possible elimination of the pathogenic cells. Therefore, we explored a strategy to enhance the capacity of MSC to kill activated immune cells by the overexpression of a death ligand. MSC were transduced with an adenovirus serotype 5 vector that is effective in gene delivery to slow cycling cells. In addition, the adenoviral DNA resides as episomes in the nucleus, and expression of the transgene is limited in time (for approximately three weeks), resulting in transient expression that is sensitive to cell-silencing mechanisms . We founThe transduction of MSC to express GFP and FasL had an insignificant impact on the cell morphology, viability, proliferation and multilineage differentiation potential. Although MSC from NOD mice present a flatter morphology than the fibroblast-like appearance specific for MSC from wild-type mice, the transduction with Ad-FasL-GFP did not induce significant changes. Likewise, adenoviral transduction had no effect on the expression of characteristic phenotypic markers such as Sca-1 and CD73 and the absent expression of CD45. We chose these markers as they are strongly expressed in the MSC derived from various mouse strains ,18. ThesThe NOD-derived MSC showed immunomodulatory properties revealed by a significant decrease of the proliferation index of both CD4 and CD8 T cells starting at a ratio of 1:20 MSC to splenocytes. Moreover, the transduction of MSC with Ad-GFP did show similar suppression properties as the naïve MSC on both CD4 and CD8 T cells at any of the ratios of MSC:splenocytes that we assessed.FasL is a common executioner of apoptosis in the immune system. FasL could induce the death of activated B and T lymphocytes only in a membrane-bound form when its interaction with its cognate receptor, Fas, initiates the intracellular signaling cascade of apoptosis. The functionality of the transduced MSC to overexpress FasL was verified by the induction of Fas/FasL-induced apoptosis upon co-incubation with A20 cells, a mouse B lymphoma cell line that expresses Fas. Additionally, we assessed the induction of apoptosis of A20 cells after exposure to MSC transduced with either the adenovirus bearing only the reporter gene, the GFP, and the one that additionally carries the FasL gene. Our results showed that the apoptosis of A20 co-incubated with MSC transduced with Ad-FasL-GFP was significantly higher than in the case of A20 co-incubated with naïve and Ad-GFP-transduced MSC.Yu and collaborators showed that increasing the FasL expression by knocking down microRNA Let-7a in bone marrow-derived MSC could induce CD3 T-cell apoptosis, both in vitro and in vivo, these cells proving more potent in the treatment of graft versus host disease and experimental colitis in murine models . To testTaken together, our data demonstrate that the NOD-sourced MSC that are modified via the adenoviral transfer of FasL are potent “killer” MSC exhibiting higher immunosuppressive potential without the loss of their main defining features. These results suggest that the FasL-overexpressing MSC could be a novel therapeutic tool to be employed in the fight with autoimmune disease like, for example, type 1 diabetes. Future research focused on identifying the optimal site of delivery of “killer” MSC, as well as the frequency of treatment administration, will define the outcome of the treatment.The NOD/ShiLtJ (NOD) mice were purchased from The Jackson Laboratory and grown in our animal facility under specific pathogen-free conditions. All animals had access to food and water ad libitum. Blood glucose of NOD females was monitored in tail blood samples at weekly intervals using a glucometer  starting at 10 weeks of age. Diabetes was defined as two consecutive blood glucose measurements above 200 mg/dL. All the procedures were conducted in accordance with the European Guidelines for Animal Welfare and were approved by the Institutional Ethical Committee of the Institute of Cellular Biology and Pathology “Nicolae Simionescu”, from Bucharest, Romania and by the National Sanitary Veterinary and Food Safety Authority (authorization no. 296/23.08.2016).6 cells/cm2 cell density. Cells were left to adhere for 24 h; then, media was replaced every 2 to 3 days. The adherent cells were left to reach confluency and then subjected to several passages, each time at a 1:4 split. Cells were grown in a 37 °C, 5% CO2 humidified incubator. All the MSC in this study were used between passage 5 and 9.MSC were isolated from bone marrow extracted from femurs and tibias of 8-week-old NOD male mice. After being passed through a 40-µm strainer, the cell aspirate was pelleted and resuspended in MSC media (DMEM low-glucose (LG) 1-g/L glucose , 10% fetal bovine serum (FBS) suitable for MSC culture  and 1% penicillin/streptomycin/amphotericin  and cultured starting with a 2 × 106 cells/mL. Maximum 200-µL cell suspension (5 × 105 cells) was added in a 1.5-mL low-retention Eppendorf tube, followed by the addition of the corresponding amount of virus. The tubes were then stirred at 1000 rpm for 45 min at 37 °C on a thermomixer, without allowing the cells to sediment. Cells were seeded at corresponding densities, depending on the following experiments.Transduction of MSC was optimized for a smaller amount of virus to infect a larger number of cells. To achieve that, MSC were detached by 0.125% trypsin-Ethylenediaminetetraacetic acid (EDTA) , washed and resuspended in DMEM 1-g/L glucose without sodium bicarbonate  supplemented with 10% MSC-FBS at a density not larger than 2.5 × 102 in 24-well plates and left to adhere overnight. Twenty-four hours after transduction, media was changed with adipogenic and osteogenic induction media, respectively, as well as normal MSC media. Media was changed every 2 to 3 days. The adipogenic induction media contained DMEM 1-g/L glucose supplemented with 10% MSC-FBS, 10−6-M dexamethasone, 100-mM indomethacin and 1% insulin-transferrin-selenium , and the osteogenic induction media was based on DMEM 1-g/L glucose supplemented with 10% MSC-FBS, 10−7-M dexamethasone, 10-mM β-glycerophosphate and 0.3-mM ascorbic acid. After 2 weeks, cells were washed with phosphate buffer saline (PBS), fixed with 4% paraformaldehyde and stained with Oil Red O to visualize the lipid droplets accumulated in the differentiated adipocytes or with von Kossa stain represented by incubation in 5% AgNO3, followed by a short 2-min rinse with 5% sodium thiosulphate to visualize the Ca2+ crystals. All the images were taken using an Olympus CKX41 microscope with an Olympus XC30 camera and minimally processed using Adobe Photoshop software.To determine the multipotency of MSC overexpressing FasL, cells transduced as described above and were seeded at a 5000 cell/cmPE, anti-mouse CD73PE/Cy7, anti-mouse CD117APC (c-kit), anti-mouse CD45PE, anti-mouse CD44PE, anti-mouse CD95PE , goat anti-mouse CD105 and the secondary rabbit anti-goatFITC . The antibody mix that was used to label the splenocytes contained anti-mouse CD45PE, anti-mouse CD4BV784 and anti-mouse CD8aAPCfire . FasL was detected using anti-FasLAF647 antibody . For all the antibodies, we used the corresponding isotypes from the same sources. Samples were analyzed using a Cytoflex flow cytometer  and CytExpert software. Determination of the proliferation index of the splenocytes was done based on the carboxyfluorescein succinimidyl ester (CFSE) readings that were processed using ModFit LTTM software . For the CFSE analysis, the unstimulated splenocytes were used as the parent .For flow cytometric analysis, MSC were stained with the following antibodies: anti-mouse Sca1Detection of FasL-induced apoptosis and cell death was done upon coculturing MSC with A20 and with freshly isolated splenocytes. The two methods will be detailed below.PB450  and analyzed by flow cytometry. We gated cells on the CD45-positive population and evaluated the percent of viable cells by excluding the cells stained with Zombie NIR (dead cells) and Annexin V (apoptotic).MSC were transduced as described above and left to adhere in MSC media. Twenty-four hours later, media was removed, and A20 cells were added in a ratio of 1:1 and incubated for another 24 h. After this time, all the cells were collected; stained with anti-CD45 antibody, Zombie NIR and Annexin VAPC and analyzed by flow cytometry. SUPERFASLIGAND® (SuperFasL) protein  was used as a control for FasL-induced apoptosis.NOD female mice, characterized by two different glycemia readings above 200 mg/dL within a week, were the source of the splenocytes. Freshly harvested cells were resuspended in RPMI medium supplemented with 10% FBS, 1% PSA and 50-µM β-mercaptoethanol (MLR medium) in the presence or absence of CD3/CD28 stimulation beads (in a ratio of 1:1 beads-to-splenocytes) . Splenocytes both unstimulated and stimulated were stained with 2.5-μM CFSE  and were cultured alone or in the presence of naïve, Ad-GFP- and Ad-FasL-GFP-transduced MSC (as above) in a ratio of 1:2 MSC-to-splenocytes for 72h in the MLR medium. At the end of the coincubation, splenocytes were carefully detached from the MSC by gentle rinsing using a 0.05-mM EDTA in PBS solution stained with anti-CD45, -CD4 and -CD8 antibodies; PI/ 7-Amino Actinomycin D (7AAD) and Annexin VThe impact of GFP or FasL expression on MSC proliferation was assessed by using an xCelligence Real-Time Cell Analysis (RTCA) DP instrument . Twenty-four hours after transduction with Ad-GFP and Ad-FasL-GFP, naïve or transduced MSC were plated in a microplate and monitored for 72 h. Doubling time was determined using RTCA software.p-value < 0.05 were considered statistically significant, and when p was not indicated, samples were not significantly different.Analysis of the data was performed using the GraphPad Prism program . Data are presented as ± SEM or SD and analyzed with the statistical tests indicated in the text and in the figure legends. Samples that had a


This study investigated the developmental paths in the use of audiovisual information for the perception of emotions and phonemes by Japanese speakers. Children aged 5 to 12 years and adults aged 30 to 39 years engaged in an emotion perception task in which speakers expressed their emotions through their faces and voices, and a phoneme perception task using phonemic information in speakers’ lip movements and speech sounds. Results indicated that Japanese children’s judgement of emotions by using auditory information increased with increasing age, whereas the use of audiovisual information for judging phonemes remained constant with increasing age. Moreover, adults were affected by visual information more than children. We discuss whether these differences in developmental patterns are due to differential integration processes for information indicative of emotions and phonemes, as well as possible cultural / linguistic reasons for these differences. Human beings use visual and auditory information to understand others’ intentions during face-to-face communication. It is well known that both emotions and phonemes are perceived through multisensory inputs. The face and the voice convey the speakers’ emotions and integrating these cues enables us to recognize the emotions of others . Recent Phonemes are perceived through lip movements as well as speech sounds. The McGurk effect  is a welIt has been suggested that Japanese people tend to be less influenced by visual information and put more weight on auditory information than Western people in the perception of emotions and phonemes –5. This We focus in the present study on the development between 5 to 12 years old. Previous developmental studies have suggested that children’s perception style develops during their school-age years. Sekiyama and Burnham’s study  showed tThe protocol of this study is approved by Tokyo Woman’s Christian University Research Ethics Committee. All the adult participants and the child participants’ parents or guardians were informed about the purpose of the study and gave written informed consent in accordance with the Declaration of Helsinki.n = 82 [34 girls], Mage = 5.6 years; SD = 0.5), 7-8-year-old , 9-10-year-old , and 11-12-year-old  children, as well as 30 to 39-year-old adults . None of adult participants had raised their own children. All participants were native Japanese speakers recruited at the National Museum of Emerging Science and Innovation (Miraikan) in Tokyo, Japan.The participants were 5-6-year-old . Participants were seated in front of the laptop computer at a distance of approximately 50 cm. The visual stimuli were displayed in the middle of the monitor at a 640×480-pixel size . Sound stimuli were presented through headphones  at a maximum volume of approximately 70 dB SPL, which was adjusted to mask the background noise in the laboratory by using amplifiers in the headphone .Participants conducted emotion perception and phoneme perception tasks in a laboratory of Miraikan. In both tasks, they were presented with audiovisually congruent and incongruent stimuli as follows.Hai, moshimoshi” (Hello), “Sayonara” (Good-bye), “Korenani” (What is this?), and “Sounandesuka” (Is that so?). Congruent stimuli were original recorded video clips, while to create the incongruent stimuli, angry (happy) voice sounds were dubbed onto happy (angry) face video clips. A total of 32 movies , angry face and happy voice, happy face and angry voice (incongruent)) × four utterances) were used as test stimuli. The average duration of the test stimuli was 2017 ms (range: 1602–2436 ms). In addition to them, two other movies were used as practice stimuli. In each trial, a fixation point was displayed at the center of the monitor for 500 ms, simultaneously with an auditory signal (440 Hz pure tone lasting 100 ms). After 500ms from the onset of the fixation, the movie, and a blank display were presented successively. Participants were asked to judge whether the woman was happy or angry and respond by pressing the D or K keys of the keyboard. The next test trial began 500 ms after the participant’s response. Following two practice trials, a total of 32 test trials that included 16 congruent and 16 incongruent trials was conducted. The order of test trials was randomized.The audiovisual stimuli of the emotion perception task were chosen from stimuli of Tanaka et al ‘s study  with a mThe phoneme perception task was conducted after the emotion perception task. To minimize the variance of the data within each task, we kept the task order constant.M = 24.7 years old, range: 22–28 years old) when the video clips were recorded, and they did not participate in the recording of the stimuli of the emotion perception task. Test stimuli contained twelve congruent movies (lip movement and sound were congruent) and six McGurk type incongruent movies (/ka/ lip movement combined with /pa/ sound). As with the emotion perception task, congruent stimuli were original recorded video clips, while in the incongruent stimuli, /pa/ voice sounds were dubbed onto /ka/ mouth video clips. The average duration of the stimuli was 996 ms (range: 834–1235 ms).The audiovisual stimuli of the phoneme perception task were previously used in Tanaka, Asakawa and Imai’s study . These aIn our preliminary experiment with adults, only small visual influences were observed when the fixation point was presented for 500 ms as in the emotion perception task, suggesting that this duration was insufficient for the phoneme perception task. To avoid a floor effect on visual influences, we set the duration of the presentation of a fixation point and the blank in the phoneme perception task longer so that the children could move their eye gaze around the fixation before the onset of visual stimuli and examine the audiovisual phoneme perception at a more appropriate length.A fixation point was displayed at the center of the monitor for 800 ms in each trial, simultaneously with an auditory signal (440 Hz pure tone lasting 100 ms), then a blank display was presented for 500 ms. After 1300ms from the onset of the fixation, the movie, and a blank display were presented successively. Participants were asked to judge whether the speaker said /ka/, /pa/, or /ta/ and respond by pressing Z, V or M keys. The next test trial began after 500 ms from the participant’s response. Each congruent movie was presented once , whereas each McGurk type movie was presented twice , resulting in a total of 24 trials. The order of test trials was randomized.F = 11.57, p < .001, ηp2 = .11). The post hoc analysis  revealed that 5- to 6-year-olds made significantly less auditory responses in incongruent trials than 9- to 10-year-olds (p = .002), 11- to 12-year-olds (p < .001), or adults (p = .002). Furthermore, 7- to 8-year-olds made significantly less auditory responses than 9- to 10- year-olds (p = .048), 11- to 12- year-olds (p < .001), or adults (p = .035). Furthermore, the auditory responses of 9- to 10-year-olds were less than those of 11- to 12- year-olds (p = .037). The results of incongruent trials suggested that Japanese children come to judge emotions by putting more weight on information from the voice with increasing age when the emotions expressed by the face and the voice are different.The auditory responses, which indicated the participants’ rate of making judgements of emotions based on the speaker’s voice in incongruent trials is shown in F = 5.44, p < .001, ηp2 = .05). The post hoc analysis revealed that 5- to 6-year-olds made significantly less correct response rate than 7- to 8- year-olds (p = .017) and 9- to 10-year-olds (p = .032). Moreover, adults made significantly less auditory responses than 7- to 8- year-olds (p = .003), 9- to 10-year-olds (p = .003), or 11- to 12- year-olds (p = .028). These results indicated that the accuracy of emotion judgements from the face and the voice increased during childhood, but slightly decreased after adolescence. The performance under each kind of stimulus is shown in As for congruent trials, we conducted a one-way ANOVA of the correct response rates . ResultsF = 14.91, p < .001, ηp2 = .13). A simple main effect analysis showed that auditory responses were significantly different between age groups for happy face and angry voice  = 22.04, p < .001). A post hoc analysis (ps < .001) revealed that auditory responses were less frequently observed in the 5- to 6- year-olds, 7- to 8- year-olds, and 9- to 10- year-olds groups than 11- to 12- year-olds and adults. Auditory responses were also less frequent in the 5- to 6- year-olds and 7- to 8- year-olds groups than 9-to 10- year-olds. The simple main effect of Age was only marginally significant for auditory response rates for angry face and happy voice  = 2.12, p = .077). This additional analysis suggests that Japanese children come to judge emotion as angry when people speak with an angry voice even though they express happiness in their faces  mixed-factor analysis of variance (ANOVA) of auditory response rates in incongruent trials to examine the effect of the combination of facial and vocal emotions. Results indicated a significant interaction between Age and Emotion  = 9.11, p < .001, ηp2 = .09). Auditory responses of adults were less than the other groups (ps < .001) in incongruent trials, whereas differences in auditory responses among the children-groups were non-significant. These results suggested that the use of visual information about lip movements remain constant during childhood, whereas it increases after adolescence.We regarded participants’ /ka/, /ta/, /pa/ responses in incongruent trials respectively as visual, fusion, and auditory responses. The auditory responses that indicate the rates of participants’ phoneme judgement based on the speaker’s voice are shown in F = 15.81, p < .001, ηp2 = .14). Adults had more frequent fusion response rates (ps < .001) than the other groups, and no significant differences were observed among the children’s groups.Moreover, we also conducted a one-way ANOVA of fusion response rates , and fouF = 5.03, p < .001, ηp2 = .50). The post hoc analysis revealed that correct responses of 5-6-year-olds were less than those of 7-8-year-olds (p = .043), 11-12-year-olds (p = .002) or adults (p = .001). Additionally, the difference between correct response rates of 5-6-year-olds and 9-10-year-olds were marginally significant (p = .073), which indicated that phoneme judgement accuracy from the face and voice increased slightly from the of age 5–6 years when the lip movement and sound were congruent.For congruent trials, we conducted a one-way ANOVA of the correct response rates . Resultsr = .03). Moreover, no significant correlations were found between voice response rates in incongruent trials for the two tasks in each group , while linguistic factors may boost the utilization of visual information. To examine this possibility, we need to dissociate the cultural and linguistic factors. This who do not speak Japanese, or with people in other East Asian countries.There are some limitations in the present study. First, the results obtained here are limited to subjects aged 5–12 and 30–39 years old. It should be noted that adult participants in the present study (30–39 years) were older than those in Sekiyama and Burnham’s  previousSecond, as mentioned in the Methods section, we adopted the constant task order to minimize the variance within each task. Thus, we cannot rule out the possibility that the emotion perception task affects performance on the phoneme perception task . We chose the constant task order because our primary aim is to compare differences across age groups. Further study is needed directly comparing the results of the two tasks regarding the balance of modalities  by counterbalancing the task order.Third, we should note that speakers’ gender was different between the emotion and phoneme tasks. Considering that there are gender differences in facial expressions  and thatThis study demonstrated different developmental paths in the perception of emotions and phonemes by using audiovisual information, which suggested different processes of integrating such information. It is, however, possible that the perceivers’ communication habits such as fixating on the other’s face affected the perception of emotions and phonemes using audiovisual information, even though the processes were different, which is a possibility that needs to be investigated in future studies. It would also be useful to investigate cultural differences in the expression of emotions using audiovisual information in naturalistic situations and explore how cultural differences in the perception of emotions reflect differences in the expression of emotions.S1 Data(XLSX)Click here for additional data file.


Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite’s metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro–induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro–induced differentiation. Our results provide insights into the mechanisms of the parasite’s mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.Mitochondrial metabolic remodeling is a hallmark of the  In most eukaryotic cells, energy metabolism is an interplay between two energy production pathways to generate ATP: an efficient mitochondrial oxidative phosphorylation (OxPhos) and an ancient glycolytic pathway. A “textbook-like” aerobic eukaryotic cell relies mainly on cost-effective OxPhos to fulfill cellular requirements for ATP. Exceptions apply to some rapidly proliferating cells, e.g., some cancer cells exploit high rates of fermentative glycolysis, irrespective of oxygen availability . OncogenTrypanosoma brucei . At. At11]. subunits . In agresubunits  and V  [oF1-ATP synthase and thus ATP depletion upon complex IV inhibition [T. brucei IF1 (TbIF1) expression is down-regulated during the RBP6 overexpression. We therefore measured the ability of the mitochondrial proton pumps, complexes III and IV, to generate the Δψm. Utilizing safranine O dye, the RBP6OE digitonin-permeabilized cells were allowed to establish Δψm in the presence of succinate as the only electron donor. Upon the addition of KCN, the rate of mitochondrial membrane depolarization was a little bit slower in RBP6OE-induced cells compared to uninduced, while a combined treatment of KCN and oligomycin, an inhibitor of FoF1-ATP synthase, depolarized the mitochondrial membranes at the same rate. These results suggest that decreased expression of TbIF1 allows the reversal of FoF1-ATP synthase to partially maintain Δψm upon inhibition of complex IV in permeabilized cells  , which ihibition . Fig 7C ed cells . But as ed cells , the coned cells , implyinOE can be partially maintained by complex I, which contributes to Δψm by coupling NADH oxidation with reduction of ubiquinone [c–mediated respiration, suggesting that in the presence of a plentiful carbon source, the canonical pathway maintains its capacity during parasite differentiation. Indeed, in a digitonin-extracted mitochondrial sample, succinate was able to stimulate ATP production via OxPhos , we did not detect any major changes  were verified by western blot analysis , which contains proline, not glucose, as the energy source. To prevent uptake of residual glucose molecules from the FBS, the RBP6OE cells were grown in the presence of N-acetyl glucosamine . Because the efficacy of differentiation correlates with the expression of RBP6 [Our knowledge of molecular mechanisms driving metabolic rewiring of the e tsetse  requirede tsetse . This voe tsetse . Compare of RBP6 , our res of RBP6 . Proline of RBP6 . Upon th of RBP6 . Higher  of RBP6 . In agreT. brucei does not synthesize lipids from citrate-derived acetyl-CoA; instead, the parasite uses mitochondrial acetate for further lipogenesis and cholesterolgenesis [Enzymatic activities of all TCA cycle enzymes have been detected in PCF trypanosomes , but dirlgenesis . Therefolgenesis  and furtlgenesis  to maint2O2) are important regulators of cellular homeostasis and play an essential role in cellular stress signaling, cell survival, differentiation, proliferation, and oncogenic transformation [2O2 by superoxide dismutases (SODs). In RBP6OE cells, a significant increase in mitochondrial ROS production coincides with elevated expression of AOX and with rechanneling of electrons entering ETC to this oxidase. This observation, interestingly, contradicts the available literature on AOX expression in other systems. For example, the introduction of Ciona intestinalis AOX into mammalian cells with severe mitochondrial dysfunction minimized ROS production by bypassing complexes III and IV [ROS  pool status [OE will remain elusive.As electrons are redirected away from complex III, during RBP6l status , or by rl status . While dT. brucei–related parasite Leishmania [Leishmania promastigotes differentiate to amastigotes. Intriguingly, ROS-generating drug menadione or H2O2 alone triggered promastigote differentiation to fully infective amastigote [T. brucei to establish infection in the midgut of the tsetse fly [2O2 molecules are prominent candidates for ROS signaling as they are long-lived, membrane-permeable, and induce reversible oxidation of thiols present in redox-sensitive proteins [2O2 signal was shown to attenuate global protein synthesis by modulating the redox status of proteins involved in translation [T. brucei metacyclic cells, which are quiescent . MultipG0 phase , and inas to PCF .T. brucei cells undergoing an 8-day-long in vitro differentiation to metacyclic cells. Even though RBP6OE is genetically induced and thus an artificial route to the differentiation of the midgut PCF trypomastigote, our–omics and functional data constitute a unique set of resources to facilitate further interrogation of intrinsic and extrinsic signaling pathways to gain deeper insights into the differentiation processes underlying not only rewiring of mitochondrial metabolism and its consequences but also changes in gene expression of surface proteins, kDNA repositioning, and mitochondrial cristae remodeling.In summary, we have provided a global transcriptomic, proteomic, and metabolomic study of T. brucei at different stages (1 × 108 cells/replicate) using the miRNeasy Kit  according to the manufacturer's protocol. An additional DNase1 digestion step was performed to ensure that the samples were not contaminated with genomic DNA. RNA purity was assessed using the Agilent 2100 Bioanalyzer . Next generation sequencing (NGS) library prep was performed with TruSeq Strand-Specific mRNA Library Prep with PolyA-Selection following Illuminas standard protocol. Libraries were profiled using the High Sensitivity DNA Kit on a 2100 Bioanalyzer and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). Libraries were sequenced on an Illumina NextSeq 500 in the Genomics Core Facility at the Institute of Molecular Biology, Mainz, Germany. The RNA-Seq measurement yielded on average 11.1 M single reads of 75 nt per sample. We assessed the quality of the sequenced reads with fastqc [https://doi.org/10.1186/s12859-016-1276-2). Spliced-leader sequences (CAATATAGTACAGAAACTGTTCTAATAATAGCGTTAGTT) were removed from the reads using cutadapt (https://doi.org/10.14806/ej.17.1.200) and then mapped to the T. brucei 11 megabase chromosomes (TriTrypDB version 36) using STAR version 2.5.2b (https://doi.org/10.1093/bioinformatics/bts635), allowing up to 2 mismatches, a minimum intron length of 21, and keeping only uniquely aligned reads . We then counted reads per gene using featureCounts (https://doi.org/10.1093/bioinformatics/btt656) from the subread package version 1.5.1 with default parameters and using the gene models provided by TriTrypDB version 36. For the differential expression analysis, we used R version 3.4.3 (http://www.R-project.org/) and DESeq2 version 1.18.1 (https://doi.org/10.1186/s13059-014-0550-8) to normalize, transform, and model the data. The counts were fitted to a Negative Binomial generalized linear model (GLM), and the Wald significance test was used to determine the differentially expressed genes between control and knockdown samples. Finally, RPKM values were calculated per gene using the library size-normalized FPM (robust counts per million mapped fragments) values from DESeq2. We applied automatic independent filtering to avoid testing genes, which were poor candidates of being differentially expressed . Differentially expressed mRNAs were identified using a threshold of Benjamini-Hochberg–corrected P values <0.05. GO enrichment analyses were performed using GO Term annotations TriTrypDB-36_TbruceiLister427_GO.gaf from TriTrypDB version 36 and Fisher’s exact test. For the comparison to the transcriptome data of the previously published dataset of procyclic and metacyclic forms of T. brucei [Total RNA was isolated from h fastqc  and dupR. brucei ,31, the T. brucei at different stages (1 × 108 cells/replicate) were washed three times in 10 mL of phosphate-buffered saline (PBS) and lysed in 6% sodium dodecyl sulfate (SDS), 300 mM DTT, and 150 mM Tris-HCl (pH 6.8), 30% glycerol, and 0.02% Bromophenol Blue. Samples were loaded on a NOVEX NuPage 4%–12% gradient gel , run for 10 minutes at 180 V, and stained with Coommassie G250. Each lane was cut and the minced gel pieces were transferred to an Eppendorf tube for destaining with 50% ethanol/50 mM ABC buffer pH 8.0. The gel pieces were dried and subsequently reduced (10 mM DTT/50 mM ABC buffer pH 8.0), alkylated (55 mM iodoacetamide/50 mM ABC buffer pH 8.0), and digested with 1 μg trypsin overnight at 37°C. The tryptic peptides were eluted from the gel pieces with pure acetonitrile and stored on a StageTip [StageTip .The proteomic measurement was performed on a Q Exactive Plus mass spectrometer  with an online-mounted C18-packed capillary column  by eluting along a 225-minute gradient of 2% to 40% acetonitrile using an EasyLC 1000 uHPLC system . The mass spectrometer was operated with a top10 data-dependent acquisition (DDA) mode.P values were corrected using the Benjamini-Hochberg method.Data analysis was performed in MaxQuant  version T. brucei PCF cells strains 29.13, transgenic for T7 RNA polymerase and the tetracycline repressor [OE cells were adapted to grow in SDM-80 medium containing no glucose and further supplemented with 50 mM N-acetyl glucose-amine to block uptake of residual glucose molecules from 10% FBS. The induction of ectopically expressed RBP6 protein was triggered by the addition of 10 μg/mL of tetracycline into the medium. Cell densities were measured using the Z2 Cell Counter . Throughout the analyses, cells were maintained in the exponential mid-log growth phase (between 2 × 106 and 1 × 107 cells/mL). The RBP6OE_catalase cell line was generated using pT7v5 plasmid containing a C. fasciculata catalase ORF sequence (cCAT TriTrypDB gene ID = CFAC1_250006200) [OE cells. The activity of the catalase was detected using a simple visual activity test. A total of 5 × 107 parasites were resuspended in 100 μL of PBS and placed on a microscopic slide. A volume of 20 μL of 3% H2O2 was added to the cells, mixed, and the formation of oxygen (bubbles formation) was monitored by eye.epressor , were gr0006200) . The pT78 cells were isolated by hypotonic cell lysis, and mitochondria were resuspended in a buffer  and lysed for one hour on ice with 2% dodecylmaltosid. The samples were spun down at 16,000g for 30 minutes and the cleared lysate protein concentrations were determined by a BCA assay. Mitochondrial lysate (20 μg) was mixed with a loading dye . After electrophoresis , the resolved mitochondrial lysates were transferred onto a PVDF membrane and probed with selected antibodies, or the native gels were directly used for in-gel activity staining of the respiratory complexes. Specific staining of complex III was achieved by incubating the gel in complex III assay buffer  by slow agitation overnight. Complex IV staining was achieved using a reaction buffer  overnight. Complex V was visualized using ATPase reaction buffer 2, 11 mM ATP) for overnight incubation by slow agitation. Mitochondrial samples for complex II detection were treated with loading dye  and they were run on 3%–12% gradient high-resolution clear-native gels (hrCNE). Specific staining was achieved by incubating the gel in staining solution: 5 mM Tris HCl 7.4, 20 mM sodium succinate, 0.2 mM phenazine methasulfate, and 2.5 mg/mL nitrotetrazolium blue chloride.BNE of mitochondria lysed with dodecylmaltoside, followed by in-gel activity staining, was adapted from published protocols . BrieflyE. coli expression plasmid, pSKB3. The proteins were overexpressed in BL21 Escherichia coli cells and purified under native or denatured conditions. Antigens were sent to Davids Biotechnologie  for pAb production. Primary antibodies used in this study were as follows: mAb anti-mitochondrial hsp70  [1-ATPase  [Protein samples were separated on SDS-PAGE, blotted onto a PVDF membrane , and probed with the appropriate monoclonal antibody (mAb) or polyclonal antibody (pAb). This was followed by incubation with a secondary HRP-conjugated anti-rabbit or anti-mouse antibody . Proteins were visualized using the Pierce ECL system on a ChemiDoc instrument ). The PageRuler prestained protein standard  was used to determine the size of the detected bands. Several antibodies  were prepared for the purpose of this study. The open reading frames of the respective genes without their predicted mitochondrial localization signal were cloned into the 1:2,000) , pAb ant1:2,000) , pAb ant1:2,000) , pAb ant1:2,000) , anti-SD1:2,000) , pAb ant1:2,000) , pAb ant1:2,000) , pAb ant2DCFHDA) and MitoSOX Red Mitochondrial Superoxide dyes , respectively. Cells in the exponential growth phase were treated with 10 μM H2DCFHDA or with 5 μM MitoSOX for 30 minutes at 27°C. A total of 1 × 107 cells were pelleted , washed with 1 mL of PBS (pH 7.4), resuspended in 2 mL of PBS, and immediately analyzed by flow cytometry . The Δψm was determined using the red-fluorescent stain tetramethylrhodamine ethyl ester TMRE . Cells in the exponential growth phase were stained with 60 nM of the dye for 30 minutes at 27°C. Cells were pelleted , resuspended in 2 mL of PBS (pH 7.4), and immediately analyzed by flow cytometry (BD FACS Canto II Instrument). Treatment with the protonophore FCCP (20 μM) was used as a control for mitochondrial membrane depolarization. To evaluate the effect of oligomycin and KCN on Δψm, the cells were incubated with 2.5 ug/mL of oligomycin or 0.5 mM of KCN in the presence of TMRE for 30 minutes at 27°C. For all samples, 10,000 events were collected. Data were evaluated using BD FACSDiva software .The cellular and mitochondrial ROS levels were determined using the 2′,7′-dichlorofluorescein diacetate (HT. brucei PCF cells (2 × 107 cells/mL) were resuspended in a reaction buffer containing the following: 200 mM sucrose, 10 mM HEPES-Na (pH 7.0), 2 mM succinate, 1 mM MgCl2, and 1 mM EGTA. The reaction was induced with digitonin (40 μM), while NaCN (1 mM) and FCCP (5 μM) were injected at specific time points throughout the assay. Changes in the amount of fluorescence over time were detected on an Infinite M200 microplate reader (TECAN) . Values were normalized according to the following equation: normalized (Ei) = Ei − Emin/Emax − Emin .Estimation of the Δψm in situ was performed spectrofluorometrically using the indicating dye safranine O . 7 cells per Oroboros O2K oxygraph chamber. The endogenous respiration of cells, as well as the substrate-induced respiration, were measured in MiR05 medium . The following respiratory substrates were used: 10 mM succinate, 5 mM proline, or 10 mM glycerol-3-phosphate. The SHAM-sensitive respiration was determined by injecting 250 μM SHAM, while the KCN sensitive respiration was assessed with 1 mM KCN. If needed, the cells were permeabilized with 4 ug of digitonin .The oxygen consumption rate was assessed at 27°C using 2 × 10OE cells were obtained by digitonin extraction. ATP production in these samples was induced by the addition of 5 mM of indicated substrates  and 67 μM ADP. The mitochondrial preparations were preincubated for 10 minutes on ice with the inhibitor malonate (6.7 mM) or KCN (1 mM). The concentration of ATP was determined by a luminometer  using the ATP Bioluminescence assay kit CLS II .ATP production was measured as described . Briefly6) were resuspended in 80 μL media and supplemented with 20 μL of Dextran, Alexa Fluor 568; 10,000 Mw . After 1 hour, the cells were fixed by formaldehyde, applied to the polylysine-coated coverslip. The coverslips were then mounted on a glass slide with ProLong Gold Antifade Mountant . Images were taken with the fluorescent microscope  with a CCD camera (Olympus DP73). To determine cells by their morphology, 100 cells were counted and assigned to a certain cell type based on their size, shape, position of the kinetoplast relative to the nucleus, and position of the kinetoplast relative to the posterior end of the cell , a metalloprotease inhibitor, to stabilize the surface proteins 24 hours prior harvesting . Then, cthe cell .7 cells were treated with 5 mM BPS, harvested , and resuspended in 200 μL of 1× PBS pH 7.4. The cells were fixed by the addition of 7.4% formaldehyde in 1× PBS pH 7.4 for 15 minutes at RT. Subsequently, the cells were washed 3 times in 1× PBS, labeled with polyclonal rabbit anti-BARP (1:400) and anti-procyclin (1:400) in 1% BSA for 1 hour at RT, followed by staining with Alexa Fluor 488–conjugated goat α-rabbit (1:400) antibody in 1% BSA. Samples were then washed, resuspended in 1 mL of 1× PBS pH 7.4 and analyzed by flow cytometry. Data were evaluated using BD FACSDiva software.The 2 × 107 cells were used for each sample. Cells were rapidly cooled in a dry ice/ethanol bath to 4°C, centrifuged at 1,300g, 4°C for 10 minutes, washed with 1× PBS, and resuspended in extraction solvent . Following shaking for 1 hour at 4°C, samples were centrifuged at 16,000g at 4°C for 10 minutes, and the supernatant was collected and stored at −80°C. The analysis was performed using separation on 150 × 4.6 mm ZIC-pHILIC  on Dionex UltiMate 3000 RSLC  followed by mass detection on an Orbitrap Fusion mass spectrometer  at Glasgow Polyomics.For the LC-MS metabolomic analysis, the sample extraction was performed as described previously ,82. Briet test. A P value of less than 0.05 was considered statistically significant. Quantitative mass spectrometry experiments were performed in four biological replicates.The number of replicates, controls, and statistical tests are in accordance with published studies employing comparable techniques and are generally accepted in the field. Statistical differences were analyzed with Prism software . Comparisons of two groups were calculated with two-tailed paired S1 FigPCA, principal component analysis; RBP6, RNA binding protein 6.(TIF)Click here for additional data file.S2 FigP values smaller than 0.05) are highlighted in red. RBP6, RNA binding protein 6.The genes with fold change larger than 2 or smaller than 0.5 (with Benjamini-Hochberg–corrected (PDF)Click here for additional data file.S3 FigThe Pearson correlation values reflect the similarity of the transcriptomes of the respective time points to the two trypomastigote types—procyclic and metacyclic forms. RBP6, RNA binding protein 6.(PNG)Click here for additional data file.S4 FigThe heatmap encompassing 5,227 z-scored LFQ quantified protein groups illustrates significant proteome remodeling during RBP6-induced differentiation. LFQ, label-free quantification; RBP6, RNA binding protein 6.(PDF)Click here for additional data file.S5 FigPCA of the proteomic samples shows the reproducibility of replicates. PCA, principal component analysis.(PDF)Click here for additional data file.S6 FigThe color key differs for each map and is always located below the heatmap. LFQ, label-free quantification; RBP6, RNA binding protein 6.(JPG)Click here for additional data file.S7 Fig2 flux per cell (right y-axis). Inhibition of AOX-mediated respiration was induced by addition of SHAM. The addition of KCN inhibited respiration via complex IV. AOX, alternative oxidase; KCN, potassium cyanide; RBP6, RNA binding protein 6; SHAM, salicylhydroxamic acid.The black lines show a decreasing concentration of oxygen in the buffer (left y-axis), while the red line shows O(PDF)Click here for additional data file.S8 FigThe color key differs for each map and is located below the heatmap. LFQ, label-free quantification; RBP6, RNA binding protein 6.(PDF)Click here for additional data file.S1 TableT. brucei strain 427 (https://tritrypdb.org/tritrypdb/), their respective best orthologs from T. brucei strain 927, and RPKM values for each sample. The experiment was performed in quadruplicates for time points 0, 2, 3, 4, 6, and 8 days upon RBP6 induction. Analyses using R version 3.4.3 and DESeq2 version 1.18.1 were used to identify differentially expressed mRNAs, which were identified using a threshold of Benjamini-Hochberg–corrected P values <0.05. RBP6, RNA binding protein 6; RPKM, reads per kilobase of transcript, per million mapped reads.Sheet 1 contains gene IDs for (XLSX)Click here for additional data file.S2 TableGene IDs belonging to four different clusters from time-course expression profiling based on K-medoids. GO enrichment analyses performed using GO Term annotations TriTrypDB-36_TbruceiLister427_GO.gaf from TriTrypDB version 36 and Fisher’s exact test. GO, Gene Ontology.(XLSX)Click here for additional data file.S3 TableT. brucei strain 427 (https://tritrypdb.org/tritrypdb/), their respective best orthologs from T. brucei strain 927, log2 fold change, Benjamini-Hochberg–corrected P values, and RPKM values for each sample. RBP6, RNA binding protein 6; RPKM, reads per kilobase of transcript, per million mapped reads.Sheets contains gene IDs for (XLSX)Click here for additional data file.S4 TableSheet 1 contains Tb427 and Tb927 gene IDs and descriptions for 5,227 protein groups identified by a minimum of 2 peptides (1 unique) and present in at least two out of four replicates. Other sheets contain protein groups differentially expressed . RBP6, RNA binding protein 6.(XLSX)Click here for additional data file.S5 TableGene IDs belonging to six different clusters from time-course expression profiling based on K-medoids. GO enrichment analyses performed using GO Term annotations TriTrypDB-36_TbruceiLister427_GO.gaf from TriTrypDB version 36 and Fisher’s exact test. GO, Gene Ontology.(XLSX)Click here for additional data file.S6 TableLC-MS metabolomic data. LC-MS, liquid chromatography–mass spectrometry; RBP6, RNA binding protein 6.(XLSX)Click here for additional data file.S1 Video7 parasites were resuspended in 100 μL of PBS and placed on a microscopic slide. A total of 20 μL of 3% H2O2 was added to the cells, mixed, and the formation of oxygen (bubbles formation) was monitored visually. PBS, phosphate-buffered saline.The activity of the catalase was detected using a simple visual activity test. A total of 5 × 10(MP4)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file. 6 Dec 2019Dear Dr Zíková, Thank you for submitting your manuscript entitled "Cell-based and multi-omics profiling reveals dynamic metabolic repurposing of mitochondria to drive developmental progression of Trypanosoma brucei" for consideration as a Research Article by PLOS Biology.Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review.However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.Please re-submit your manuscript within two working days, i.e. by Dec 08 2019 11:59PM.https://www.editorialmanager.com/pbiologyLogin to Editorial Manager here: http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit. During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review. plosbiology@plos.org if you have any queries relating to your submission.Feel free to email us at Kind regards,Lauren A Richardson, Ph.DSenior EditorPLOS Biology 10 Jan 2020Dear Dr Zíková,Thank you very much for submitting your manuscript "Cell-based and multi-omics profiling reveals dynamic metabolic repurposing of mitochondria to drive developmental progression of Trypanosoma brucei" for consideration as a Research Article at PLOS Biology. As you will read, the reviewers appreciated many aspects of your work. However, they do raise concerns that will need to be addressed in a revision. Of particular note, the reviewers note missing controls and discrepancies in the data that need to be investigated further. After discussion with the Academic Editor, we have decided that your manuscript would be better suited for further consideration as a 'Methods and Resources' article. During your revision, please highlight the value of these datasets for further studies and make them as accessible as possible. Lastly, the Academic Editor notes that in lines 183-187 it is stated that “cluster 1 includes proteins that are being upregulated…in agreement with the transcriptomic data, it contains enzymes involved in energy metabolism….” but that this is not reflected in the figure.In light of the reviews (below), we will not be able to accept the current version of the manuscript, but we would welcome re-submission of a much-revised version that takes into account the reviewers' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent for further evaluation by the reviewers.We expect to receive your revised manuscript within 2 months. plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.Please email us  and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/*PLOS Data Policy*http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information . For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5Please note that as a condition of publication PLOS' data policy  such that it can be cited independently in the future. For instructions see: Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Lauren A Richardson, Ph.DSenior EditorPLOS Biology*****************************************************REVIEWS:Reviewer #1: Following uptake into the tsetse fly, African trypanosomes multiply as procyclic cells. After differentiation into epimastigote forms, infectious trypomastigotes are formed which are again transmitted to the mammalian host, closing the digenetic life cycle. The work submitted by Dolezelova et al. takes advantage of a previous finding that overexpression of RBP6, a specific RNA-binding protein, induces the differentiation process in vitro. The authors present thorough transcriptome, proteome and metabolome analyses with the aim to get a deeper insight in the changes occurring during the differentiation processes in the insect vector of Trypanosoma brucei. Most remarkably, they show that ectopic expression of catalase in the cytosol of procyclic parasites halts the in vitro-induced differentiation and suggest that hydrogen peroxide may act as signaling molecule.The data presented are new and of significant interest especially for scientists working on the differentiation of African trypanosomes and related parasites. However, the manuscript requires a number of corrections and alterations. Major points:How long where the cells grown in the absence of glucose and presence of N-acetyl glucosamine before RBP6 overexpression was induced? Can the authors exclude that non-differentiating procyclic cells show similar alterations  when they are long-term cultured under these conditions? Non-induced cells cultured for six or eight days in the absence of glucose but presence of N-acetyl glucosamine should have been included for control.Figure 5, why is the O2-flux at day 0 in (A) resting cells as high as in (B) glycerol-3-phosphate-induced cells? Figure 6 and text, lines 265-269, please clarify the section and remove redundancies. The Western blots of complex IV at day 4 do not show any significant changes; lines 270-275, the description refers exclusively to day 2. Please comment why at day 6, the activity of complex II is again as low as on day 0.Figure 8F, please explain the extremely high ADP/ATP ratio of 2, even on day 0. Usually, non-stressed cells have an ADP/ATP ratio of 0.1 to 0.2. Figure 10, C, please include the number of experiments and describe the Western blot in the legend, including the time point at which the samples were taken; D, please include the number of experiments; E and text, line 387, the statement about the relative AOX expression is not convincing as in the RBP6-OE-catalase sample (left blot), the AOX level in bloodstream cells is higher than in the RBP6-OE sample (right blot); and line 389, why are the aconitase levels in the catalase-expressing cells lower than in the cells not expressing catalase?; lines 392-397, the section is very speculative, especially " including signaling molecules" and should be modified; F, also in RBP6-OE cells without catalase , the TMRE fluorescence increases upon induction; G and B, please use the same scale for the y-axis. Lines 45-46, should it read "aerobic cells"? Otherwise, the sentence is not clear.Line 225, the statement that glucose-6-phosphate isomerase functions only in the gluconeogenesis direction is not correct. Please define SBPase.Lines 296-298, the sentence starting with "Accordingly" is not clear. Minor points:For readers not specialized in the omics-field, the methodology should be described in more detail and the abbreviations  defined in the text and/or figure legends. The same abbreviation should be used throughout the manuscript . "Oxal" should be replaced by "oxaloacetate" etc.Please replace in line 36, "acted" by "appears to act" and in line 37, "exogenous" by "ectopic".Line 183, please define the clusters 2, 5 and 6.Line 239, please replace "synthase" by "synthetase".Line 313, please include .Line 315, AOX is not described in Fig 6; should it be Fig3B?Lines 349-351, please describe more precisely which changes are consistent with the scheme in Fig 4?Line 355, the authors describe that the reduced form of trypanothione was not detected, what is with the oxidized form?Line 416, please modify the sentence. It should read "we detected lower levels of proline and glutamate which may suggest a higher consumption".Line 434, replace "synthetase" by "synthase".Line 482-484, to get a deeper insight in the subcellular origin of hydrogen peroxide, catalase could be expressed in the mitochondrial matrix.Figure 2D, it should read "Protein phosphorylation"Figure 3A, "day" and "log 2-fold", respectively, should be included in front of the numbers. Figure 4, the use of different colors for the upregulated reactions would facilitate reading. All abbreviations should be defined in the legend. Pyruvate is given twice in grey boxes, ones without, please clarify.Figure 5C, what is the meaning of s.d. for only two values?Figure 7 legend, include after "cells" "that", see also comment for Figure 5The supplementary figures S1-S5 were missing in the manuscript provided and thus were not evaluated.---------------Reviewer #2: Trypanosoma brucei lives in two hosts, mammals and tsetse flies, and adapts its metabolism to its different environments. Until recently, the majority of life-cycle stages from tsetse could not be cultured. In this study the authors exploit a system first described by Kolev et al., over-expression of the RNA-binding protein RBP6. This drives differentiation in culture from the procyclic (fly midgut) form, via a series of intermediate life-cycle stages, to the metacyclic form which is infectious for mammals. In contrast to Kolev et al., the authors use a glucose-free medium, which appears to make differentiation more efficient. This study provides time courses of the transcriptome, proteome and metabolome, with a focus on the mitochondrion which changes during development. Interestingly, reactive oxygen species seem to play a role in differentiation as expression of catalase  blocks life-cycle progression in cultures. I appreciate this work as it contains a wealth of data, but I would have liked to have seen it analyzed more extensively. The authors focus on the metabolic capability and remodeling of the mitochondrion, an organelle that is present in almost all eukaryotes. Nevertheless, for the following reasons, I am in two minds about whether it fits the scope of PLoS Biology or if the data would be more appropriate for another PLoS journal (e.g. PLoS Pathogens):1. The work sheds most light on differentiation and could identify new stages or intermediate stages in the life cycle. It might then be easier to attribute metabolic pathways to specific stages of the life cycle. 2. There are nuances in the system that are difficult for non-parasitologists to appreciate. The differentiating cultures consist of mixtures of cells, some of which do not usually coexist in one tissue. The data thus reflect these mixtures overlaid upon each other. This differs from a previous study by Cristiano et al., in which in vitro-derived metacyclics were purified prior to transcriptomic and proteomic analyses. Incidentally, their data strongly suggested that metacyclic forms and bloodstream forms are metabolically similar. Importantly, by day 8 in the current study, there is a considerable proportion of "procyclic cells" (more about this below). This means that the cells in culture are either dedifferentiating and/or that there remaining epimastigotes are non-dividing and are being overgrown by procyclics. This may also explain why the clusters of co-expressed transcripts almost all change their trend on day 8.3. Although the complex life cycle is clearly described, it is not sufficiently clear how different stages were categorized. For example, in Figure 1A, panel day 2 epimastigotes: only one cell has the classic anteronuclear kinetoplast. One cell has a procyclic configuration. The other two might be transitioning as the kinetoplast is very close to or over the nucleus. Intriguingly, if the majority of cells on day 2 are epimastigotes, they are not expressing BARP proteins . While it is known what salivary gland epimastigotes express BARPs, it is not known if they are expressed by epimastigotes in the foregut. Might there be two populations of epimastigotes in these cultures and are their mitochondria and metabolism different? The authors may be able to extract some more information from their data sets.4. The procyclic forms on day 8 do not reflect the natural life cycle. They don't seem to express GPEET procyclin protein or mRNA. What about the other procyclin marker, EP? This information could be extracted from the RNA-seq data. If they are procyclic forms, it should be made very clear that this is a culture artefact. Alternatively, it might be better to remove most of the day 8 data. 5. RNA-seq data (Table S1). The gene IDs are from the 2018 version of T. brucei 427. Although this makes sense, because this cell line is a derivative of 427, it makes it harder to browse the data when it comes to differentially expressed genes. Would it be possible to include the orthologs in T. brucei 927 and gene names in all of the tables?6. The role of ROS and the effect of ectopic catalase are intriguing. It was shown recently that trypanosomes expressing catalase were less successful at establishing midgut infections. Based on the data shown here (presence of GPEET absence of BARP), it looks like the block in differentiation occurs much earlier than the transition from epimastigotes to metacyclic forms. Analyzing the cells in these cultures by immunofluorescence or flow cytometry, in addition to morphology, would be informative. Are the majority procyclic forms (expressing GPEET) or is this a sub-population?7. The metabolome is hardly discussed apart from metabolites that change in concentration over the course of the experiment. Can more information be extracted? Are there indications of unusual pathways or missing pathways?Minor points:8. "Morphotype" is not a word that is normally applied to trypanosomes. Should this be life-cycle stage? Monoformic (monomorphic?). This refers to bloodstream forms that have been syringe-passaged until slender forms lose the capacity to differentiate to stumpy forms. 9. The word "and" is missing from line 119. It should be  … typically smaller than epimastigotes and PCF, and are ….---------------Reviewer #3: In this manuscript Doleželová et al took advantage of a previously established in vitro differentiation system based on the over-expression of the RNA binding protein 6 (RBP6) to follow changes at the RNA, protein and metabolite levels during the development of Trypanosoma brucei procyclics to infectious metacyclics. Although this development takes place in the tseste fly vector, investigations in the fly are not feasible due to difficulties in acquiring enough parasites, especially for proteomic and metabolomic studies. Thus, the RBP6 over-expression system provides an ideal model system. The authors performed a careful multi-omics study and provide convincing evidence for redirection of electron flow from the cytochrome mediated pathway to an alternative oxidase. This is a rich set of data that will be very valuable to the community. Most importantly, they not only provide insights into the mechanisms of the parasite´s mitochondrial rewiring, but also strengthen the emerging concept that mitochondria act as signaling organelles through the release of reactive oxygen species to drive cellular differentiation.There are only a few minor points that will need attention.1. A recent publication  describes an RNA-seq analysis of the time course of RBP6 induction. Although the data presented here are more detailed due to the higher efficiency of differentiation, a comparison of the results is warranted.2. The sentence starting on line 333 needs a reference. 3. Line 496: "Even though RBP6OE is not the physiological route to differentiation of the midgut PCF trypomastigote, " What do the authors mean? Do they have evidence for the physiological route to differentiation? 12 Mar 2020AttachmentResponse to the reviewers.docSubmitted filename: Click here for additional data file. 22 Apr 2020Dear Dr Zíková,Thank you for submitting your revised Methods and Resources article entitled "Cell-based and multi-omics profiling reveals dynamic metabolic repurposing of mitochondria to drive developmental progression of Trypanosoma brucei" for publication in PLOS Biology. I have now obtained advice from one of the original reviewers and have discussed these comments with the Academic Editor. Based on this review (attached below), we will probably accept this manuscript for publication, assuming that you will modify the manuscript to address the remaining points raised by the reviewers. You should discuss the remaining points and, if required, modify the manuscript. This includes the ADP/ATP issue, which remains unresolved. 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For point 4, the authors used two independent kits to determine ADP/ATP levels, which adds credence to their findings, but they avoid mentioning that the levels of ADP are unusually high by using a "normalised" ratio of 1.Points that I raised.2.Again, most have been dealt with satisfactorily. There is one discrepancy, however. In the Western blot in Figure 10E, BARP is not detectable in cells expressing catalase, but is detected in Figure 10F (flow cytometery) in about 10% of cells. This may be a sensitivity issue, but it should be addressed.3. Please check the revised version for typos, etc. There are several places where spaces are missing between words, letters were transposed or singular and plural didn't match.Media should be medium (if it is only one). "Mixture" might be more appropriate than "blend" to describe the cultures. 19 May 2020AttachmentRebuttal letter_fin_R3.docSubmitted filename: Click here for additional data file. 27 May 2020Dear Dr Zíková,On behalf of my colleagues and the Academic Editor, André Schneider, I am pleased to inform you that we will be delighted to publish your Methods and Resources in PLOS Biology. The files will now enter our production system. You will receive a copyedited version of the manuscript, along with your figures for a final review. You will be given two business days to review and approve the copyedit. Then, within a week, you will receive a PDF proof of your typeset article. You will have two days to review the PDF and make any final corrections. If there is a chance that you'll be unavailable during the copy editing/proof review period, please provide us with contact details of one of the other authors whom you nominate to handle these stages on your behalf. 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Trans-resveratrol, a well-known plant phenolic compound, has been intensively investigated due to its association with the so-called French paradox. However, despite its high pharmacological potential, trans-resveratrol has shown relatively low bioavailability. Trans-resveratrol is intensively metabolized in the intestine and liver, yielding metabolites that may be responsible for its high bioactivity. The aim of this study was to investigate and compare the metabolism of trans-resveratrol (tRes), cis-resveratrol (cRes) and dihydroresveratrol (dhRes) in an in vitro epithelial model using Caco-2 cell lines. Obtained metabolites of tRes, cRes and dhRes were analyzed by LC/MS Q-TOF, and significant differences in the metabolism of each compound were observed. The majority of tRes was transported unchanged through the Caco-2 cells, while cRes was mostly metabolized. The main metabolite of both cis- and trans-resveratrol observed as a result of colon microbial metabolism, dhRes, was metabolized almost completely, with only traces of the unchanged molecule being found. A sulphate conjugate was identified as the main metabolite of tRes in our model, while a glucuronide conjugate was the major metabolite of cRes and dhRes. Since metabolism of simple phenolics and polyphenols plays a crucial role in their bioavailability, detailed knowledge of their transformation is of high scientific value. Chromatography was carried out on a Kinetex 1.7 μm F5 100 Å 100 × 2.1 mm column . Detailed description of analysis was previously published by Jarosova et al., 2019 . The lisA is the actual concentration at the time point, CP are the previous concentrations, and CM is the concentration measured at the time point. Values are expressed as a mean ± standard deviation. Microsoft Excel, SPSS  version 25, and Statistica12  were used for basic statistical analysis and graph creation. Quantitative data were normalized to 20 µM to correct the minor dilution errors. The experiments for tRes and cRes were carried out in four biological replicates, dhRes in five biological replicates. Each of them was prepared in three technical repetitions.Because of reduction in donor concentration on the basolateral side after every sampling, the actual concentrations at each time point were counted according to following equation:The fate of tRes, cRes, and dhRes in the intestinal model significantly differed for each test compound . From thO-sulphate, tRes-3-O-glucuronide, and tRes-4′-O-glucuronide. Sulphate was the dominant metabolite of tRes. After 4 h of experiment, 3.96 ± 0.84 µM of tRes-3-O-sulphate was detected on the basolateral side, 2.75 ± 0.53 µM on the apical side, and 0.09 ± 0.02 µM in the cells, respectively. Metabolites tRes-3-O-glucuronide, and tRes-4′-O-glucuronide were detected on the basolateral side at 1.15 ± 0.15 µM and 0.39 ± 0.06 µM, respectively, and on the apical side at 0.52 ± 0.09 µM and 0.18 ± 0.05 µM, respectively. Similarly, three metabolites, cRes-sulphate and two cRes-glucuronides, were detected for cRes. However, contrary to tRes, cRes-glucuronide seemed to be the dominant metabolite of cRes. Two metabolites, dhRes-sulphate and dominant dhRes-glucuronide, were detected for dhRes. No isomeric transformations were detected for any compound during the incubation as well as during the storage of the samples.As seen in The time-dependent changes of metabolism are shown in The metabolism of natural compounds before they reach the bloodstream plays a crucial role in their bioactivity. When reaching the colon, stilbenoids are intensively metabolized by colon microbiota and transported through the enterocytes, possibly mainly in the form of conjugates. The aim of our experiment was to find the most important metabolites and compare the metabolic fate of tRes, cRes and dhRes in the intestinal epithelium using an epithelial Caco-2 cell line model. We found major differences among each of the parent compounds.O-sulphate, tRes-3-O-glucuronide, and tRes-4′-O-glucuronide, were detected. The intensity of tRes-3-O-sulphate was about three times higher than the sum of both glucuronides. A similar study [cis isomers or hydrogenated metabolites were found during the passage of tRes through Caco-2 cells.The majority of tRes 57.2%) was transported unchanged to the basolateral side; this was shown in a similar study , where a.2% was tar study  of tRes ar study , a glucuar study . This mitrans isomer. The majority of cRes was metabolized and only 32.2% passed unchanged to the basolateral side. Only 5.5% of cRes was detected on the apical side after 4 h of incubation, indicating bidirectional transport with strong predominance from the apical to the basolateral side, which is an important factor affecting its bioavailability. Similar to tRes, only traces of cRes were detected intracellularly after 4 h of incubation, indicating the efficient active transport of these compounds out of the cells. Three metabolites of cRes were detected, cRes-sulphate, and two cRes-glucuronides. In contrast to tRes, which was mainly conjugated with a sulphate, cRes was shown to conjugate mainly with glucuronic acid. The intensity of cRes glucuronides was about seven times higher than that of sulphate. The glucuronidation of tRes and cRes by Caco-2 cell lines was demonstrated in an earlier study [trans isomers or hydrogenated metabolites were found in our model.The metabolism of cRes differed significantly from its er study  where thcis or trans isomer of resveratrol. This bond arrangement allows dhRes to comply with different active site positions of UGT present in cells and it might explain its prevalent glucuronidation similar to cRes. Interestingly, dhRes-sulphate was the only metabolite detected at a higher intensity (2-fold) on the apical side than on the basolateral side and only traces of it were detected intracellularly. This indicates efficient active transport of this metabolite to the apical side, which may decrease its absorption into the blood stream and simultaneously prohibit the potentially positive effect within enterocytes. In an in vivo study in rats [Dihydroresveratrol is the main gut microbiota metabolite of resveratrol as was observed in our previous study . After a in rats , 30 min TM system using Caco-2 cell lines was explored, detecting altogether eight principal metabolites. Our results showed significant differences in the metabolism of resveratrol configurational isomers. The compounds differed in degrees of metabolism, tRes was metabolized by 20%, followed by cRes (62%) and dhRes (99%) A conjugate with sulphate was identified as the main metabolite of tRes, while a glucuronide was a major metabolite of cRes and of dhRes.In conclusion, the permeability of tRes, cRes and dhRes in a Transwell